PROJECT SUMMARY/ ABSTRACT Background: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited cardiac disorder that affects approximately 1 in 10,000 individuals. Between 50-60% of CPVT cases are autosomal dominant inherited mutations in the cardiac ryanodine receptor (RyR2), which releases Ca2+ from the sarcoplasmic reticulum (SR) in response to local increases in cytosolic Ca2+ in a process called Ca2+- induced Ca2+- release (CICR). RyR2 may also activate through increases in luminal Ca2+, in a process called store overload induced Ca2+ release (SOICR). This pathway may occur during adrenergic stimulation or from pathophysiological conditions, such as CPVT. Several FDA approved pharmaceuticals (carvedilol, flecainide, and verapamil) have been shown to decrease SOICR and arrhythmic events and have been suggested to act directly on RyR2. Recent structures for RyR1 and RyR2 have been solved using electron cryomicroscopy (cryo-EM). However, further study is needed to fully understand the mechanism behind SOICR and the luminal Ca2+ sensor of RyR2. We propose to solve the structure of RyR2 in a lipid bilayer; to examine the interactions and binding sites of carvedilol, flecainide, and verapamil; and to examine their effects on a known CPVT-causing RyR2 mutation, V4880A, which has been shown to increase luminal Ca2+ sensitivity. We hypothesize that carvedilol, flecainide, and verapamil interact directly with RyR2 to stabilize the channel in response to luminal Ca2+ activation. Methods and Specific Aims: We propose three specific aims to examine these interactions through structural and functional analyses. First, we will investigate the structure of RyR2 in a lipid bilayer, using cryo-EM and lipid nanodiscs in porcine heart. We will then determine the structural basis for interactions of carvedilol, flecainide, and verapamil with wild-type (WT) and mutant RyR2 (RyR2V4880A). Lastly, we will examine functional effects of the drugs on the WT and RyR2V4880A channels, using single channel lipid bilayer recordings and calcium imaging. Conclusions: We expect to observe a direct interaction between the pharmaceutical compounds of interest and RyR2, stabilizing the channel and reducing the probability of opening in response to luminal Ca2+. We expect to see an attenuation of the RyR2V4880A channel?s heightened sensitivity to luminal Ca2+. These experiments will provide critical insights into mechanisms of luminal Ca2+ activation during healthy and disease states. Training Component: Not only will this project serve to increase our knowledge of Ca2+ handling mechanisms during normal and pathophysiological states and aid in the development and optimization of therapeutic strategies, but it also has a significant training component. This proposal will provide the applicant with highly skilled technical training in structural biology, using a combination with the sponsor?s expertise in cryo-EM and the institution?s Imaging and Analysis facilities, while also providing quality career development training to assist in the transition from postdoctoral trainee to independent cardiovascular researcher.